Method for modifying production of fruit ripening enzyme

ABSTRACT

Process for the inhibition of the production of a gene product in a plant cell which comprises generating in the cell while the gene product is being expressed mRNA from recombinant DNA coding for part only of the gene product: also constructs for use in the process, and cells and plants that carry out the process. Specifically applicable to control of fruit ripening, in particular in tomatoes.

This is a continuation of application Ser. No. 07/621,714, filed Dec. 5,1990 now U.S. Pat. No. 5,296,376 which is a continuation-in-part of Ser.No. 07/119,614, filed Nov. 12, 1987 and a continuation of PCTapplication Ser. No. GB 90/01827, filed Nov. 26,1990, which included theU.S. as a designated filing.

This invention relates to novel DNA constructs, plant cells containingthem and plants derived therefrom. In particular it involves the use ofrecombinant DNA technology to control, and more specifically to reduceor inhibit, gene expression in plants.

Plant development is a complex physiological and biochemical processrequiring the co-ordinated expression of many genes. It produces plantswhose products, such as such as roots, foliage, fruit and seeds, areused in agriculture and food production. It has long been the aim ofscientists to develop methods which allow the manipulation of thesegenes for the purposes of producing improved plant varieties. Suchvarieties may be resistant to insects or herbicides, have improvedagronomic performance or be of better quality. For this purpose, manymethods have been developed for the isolation of plant genes, and theircharacterisation both in vitro and in vivo. The in vivo work has beensupplemented by the development of transformation techniques. Thesepermit the design of novel plant varieties, having altered and improvedphenotypes desired in the agricultural industry.

One objective which has been desirable for many years, but which hasonly recently been achieved is the inhibition of specific genes whichmay have deleterious effects on plant growth or performance or thequality of plant products. This has been done by the expression ofantisense RNA to the gene which is to be inhibited. A number of examplesare now found in the literature which demonstrate that this method canbe made to work very effectively in plants. A typical example isdisclosed in our European patent specification EP 271988 (equivalent toU.S. Ser. No. 07/119614). This shows the inhibition by antisense RNA ofenzymes, in particular polygalacturonase and pectin methylesterase,involved in cell wall modification during tomato fruit ripening.

Antisense RNA is a technique which will now find wide application in themodification of crop plants. Its mechanism is not clearly understood.One theory is that antisense RNA produced in the nucleus of transformedplants will form RNA-RNA hybrid molecules which will lead to theinhibition of the expression of the specific gene to which antisense RNAhas been expressed. An alternative plausible hypothesis is that theeffects is due to hybrids of RNA with DNA. In experiments carried out sofar, enzyme inhibition of greater than 95% has been observed in theprimary transformed plants. This level of inhibition has been increasedfurther by genetic experiments, in which the copy number of theantisense gene has been doubled. Thus inhibition of nearly 100% of PGgene expression has been achieved.

Although this method works well, there is the need for the developmentof other methods which lead to the inhibition of gene expression throughalternative molecular pathways in order to widen the repertoire oftechnical possibilities which will permit the fine-tuning of geneexpression in transgenic plants. The present invention provides such analternative method.

According to the present invention we provide a process for theinhibition of the production of a target gene product in a plant cellwhich comprises generating in the cell while the target gene is beingexpressed mRNA from recombinant DNA coding for part only of the geneproduct. We further provide novel constructs for use in the process ofthe invention which comprise recombinant DNA coding for part only of atarget gene naturally expressed in a cell which DNA is expressed underthe control of a promoter sequence operative in plant cells. Theinvention further comprises novel cells and plants in which the processis realised or which (or ancestors of which) have been transformed withthe constructs of the invention.

Inhibition of gene products according to the invention may be partial oralmost complete. It is not as yet clear why the invention works. Thealmost complete inhibition of gene expression that can be obtained undercertain circumstances is particularly surprising.

DNA constructs according to the invention preferably comprise a DNAcoding base sequence at least 50 bases in length. The upper limit to thebase sequence depends on the size of the gene whose product is to beinhibited. The theoretical upper limit will generally have to beestablished by trial and error in each case. However for most practicalpurposes it is unnecessary to establish this upper limit, as forconvenience it will generally be found suitable to use sequences between100 and 1000 bases in length. The preparation of such constructs isdescribed in more detail below. Many constructs suitable for use in theprocess of the present invention are described in U.S. Ser. No.07/119,614 (the entire disclosure of which specification is incorporatedherein by reference). In that specification they are referred to as“sense” constructs and generally given the suffix “S”.

According to the invention we propose to use both constitutive promoters(such as cauliflower mosaic virus 35S RNA) and inducible ordevelopmentally regulated promoters (such as the ripe-fruit-specificpolygalacturonase promoter) as circumstances require. Use of aconstitutive promoter will tend to affect functions in all parts of theplant in which the target gene is expressed: while by using an inducibleor tissue-specific promoter, functions may be controlled moreselectively in individual organs, tissues or cells, or at particularstages of the life cycle.

The present invention will find wide potential use in modifying plantsin useful ways. Plant gene products that may be inhibited by theinvention are of very diverse kinds. We believe that the mechanism ofinhibition is independent of the nature of the gene or gene product.Thus in principle any the production of any kind of gene product may beinhibited.

Thus we propose to apply the process of the invention to the inhibitionof gene products in all types of economically useful plants. These mayinclude for example field crops such as corn (maize), sugar beet,sorghum, and sunflower; cereals such as wheat, barley and rice; legumesand pulses such as beans and peas; grasses; trees; leafy vegetable cropssuch as cabbage, lettuce and spinach; root crops such as potato, turnip,carrot; onions; fruit of all kinds; and ornamentals such as tulips,roses, carnations and azaleas.: The gene products to be inhibited may bethose which are not desired in the crop (eg toxins) or those whosereduction can contribute to improved plant characteristics, such asyield, field performance or product quality.

By way of example only, and without any implied limitation on its fieldof use, we will describe the invention further with particular referenceto possible uses in controlling fruit ripening processes.

The plants to which the present invention can be applied includecommercially important fruit-bearing plants, for example melons,peaches, bananas, apples, strawberries, kiwi fruit, and in particularthe tomato.

In this way, plants can be generated which may have one or more of thefollowing characteristics:

Novel flavour and aroma due to changes in the concentrations and ratiosof the many aromatic compounds that contribute to fruit flavour;

Sweeter fruit (e.g. tomatoes) due to decrease in the accumulation ofacids (e.g. citric or malic acid) thereby allowing the flavour of thesugars to dominate;

Modified colour due to inhibition of the pathways of pigmentbiosynthesis (e.g. in the case of tomatoes, lycopene, β-carotene);

Longer shelf life and better storage characteristics due to reducedactivity of degradative pathways (e.g. cell wall hydrolysis);

Improved processing characteristics due to changed activity of enzymescontributing to factors such as: viscosity, solids, pH, elasticity;

Modified fruit shape, thus improving packing and storagecharacteristics;

Extended leaf biosynthetic activity due to inhibition of enzymesresponsible for the degradative processes involved in senescence (inparticular, leaf senescence); thus improving plant productivity.

Among the gene products that can be inhibited by the process of theinvention is the enzyme polygalacturonase (PG). PG is a major cell wallhydrolase expressed specifically during the ripening of fruits. Thespecific embodiment of the invention that we have so far studied mostthoroughly is that of PG expressed during the ripening of tomato fruit.

It has previously been demonstrated, that one gene encodes three PGisoenzymes which are produced by posttranslational modification of theprimary translation product. The exact nature of the modification of thedifferent isoenzymes is not known. It is also controversial, which ofthe isoenzymes (found in the middle lamellar fraction of the tomato cellwall) is the functional enzyme. The structure of the PG mRNA has beendetermined, and it has been demonstrated (by comparing the sequence ofthe protein predicted from the mRNA with that of the PG 2a isoenzymefound in the cell wall of the tomato fruit) that the protein encoded bythe mRNA contains both N- as well as C-terminal extension which havebeen postulated to be involved in transport of the protein to its siteof action. No in vivo or in vitro experiments using tomato fruit cellwalls have been carried out which describe the transport of the PGprotein to the cell wall. The experiments described below do notelucidate the mechanism by which the invention achieves the inhibitionof production of the target gene product. Various mechanisms may beenvisaged, for example interference with the cell's transcriptionmechanism by DNA-RNA basepairing, or with RNA processing. It may provethat the mechanism is basically related to that by which antisense RNAinhibits a target gene product. Whatever the mechanism, the examplesgiven below clearly show that the method of the invention, using theexpression of part of the PG cDNA, results in substantial inhibition ofPG. Tomato fruit and their seeds and progeny of these plants will finduse in the production of new tomato varieties containing reduced PG.These plants will be useful in the production of tomatoes of improvedquality, which may have a longer storage life, better transportability,better field holding (ie, fruit lasts longer in good condition on theplant prior to harvest) or be easier to process, and may produceimproved products such as whole peeled tomatoes, puree, ketchup orsauces.

Clearly the invention can be used not only for the inhibition of PG, butalso for the inhibition of other cell wall hydrolases such as pectinesterase, galactosidase, glucanase, xylanase and cellulase. It can alsobe used for the inhibition of other enzymes important for plantdevelopment and function.

The invention will now be described further with reference to theExamples and to the accompanying drawings, in which:

FIG. 1 shows the construction of an expression vector according to theinvention;

FIG. 2 shows schematically the fragments of pTOM6 and of the tomatopolygalacturonase gene clone gTOM23 used in certain of the Examples.

The following Examples 1-25 illustrate the preparation of vectorsaccording to the invention, vectors useful for making such vectors, andvectors useful for carrying out the process of the invention. TheseExamples are taken from our U.S. application Ser. No. 07/119,614 (theentire disclosure of which specification is incorporated herein byreference). Example 27 shows the effectiveness of the invention ininhibiting the production of polygalacturonase. All cloning proceduresare performed under standard conditions as described by Maniatis et al(1982) “Molecular Cloning”, Cold Spring Harbor Laboratory. Vectors forwhich an NCIB Accession number are given have been deposited at theNational Collections of Industrial and Marine Bacteria, Aberdeen,Scotland.

EXAMPLE 1 Construction of the Plasmid pJR1

A. Isolation of the nos 3′ end

10 μg of pWRECK2-CAMV-CAT-1 (NCIB Accession No.12352) was digested withPvuI in order to linearise the DNA, under conditions recommended by themanufacturer. The completeness of digestion was analysed by running analiquot of the reaction of 0.8% agarose gels. The reaction was stoppedby extraction with phenol/chloroform. DNA was precipitated with ethanoland dried under vacuum. The cohesive ends were removed by incubation ofthe linearised DNA with T4 polymerase at 37° C. for 30 minutes. Theenzyme was inactivated by incubation at 65° C. for 15 minutes. Thereaction volume was increased by the addition of HindIII buffer andHindIII enzyme was added. The reaction was carried out for 2 hours at37° C. The 250 bp PvuI/HindIII fragment wasisolated from agarose gels byelectroelution. DNA was phenol/chloroform extracted, precipitated withethanol and resuspended in water.

B. Removal of the CaMV 3′ end from pDH51

2μg of pDH51 (Pietrzak et al, (1986) Nucleic Acids Research 14,5857-5868) was digested with SphI at 37° C. for 2 hours under standardconditions. The reaction was stopped by extraction withphenol/chloroform. DNA was precipitated with ethanol and resuspended inwater. Cohesive ends were removed by treatment with T4 polymerase for 30minutes at 37° C. The buffer volume was increased and HindIII was added.The mixture was incubated for 2 hours at 37° C. The resulting 3.2 Kbfragment was isolated after gel electrophoresis on agarose gels byelectroelution. The DNA was extracted with phenol and chloroform,precipitated with ethanol and resuspended in water.

C. Cloning of nos 3′ end into pDH51 to give pJR1

1 μg of pDH51 prepared under (B) was ligated with 100 ng of nos 3′ endprepared under (A) in a total of 15 μg in the presence of T4 ligase.Incubation was carried our for 24 hours in 16° C. An aliquot of theligation was transformed into competent TG2 cells. An aliquot of thetransformation mix was plated onto ampicillin and Xgal containingL-plates. White colonies were picked, and the DNA examined byrestriction analysis. Molecules containing the nos 3′ end werecharacterised by the presence of a 260 base pair HindIII-BamH1 fragment.These plasmids were called pJR1

EXAMPLE 2 Construction of Plasmids pDHC1 and pDHC4

A. Isolation of a 730 bp HinfI fragment from pTOM6

5 μg pTOM6 (NCIB Accession No 12351) was treated with Hinf1 for 2 hoursat 39° C. under standard conditions. The 730 bp Hinf1 fragment wasisolated after separation on agarose GELS. The cohesive ends of thisfragment were filled in with DNA polymerase Klenow fragment A. The DNAwas phenol extracted and ethanol precipitated.

B. Linearisation of pDH51

1 μg pDH51 was treated with SmaI for 2 hours at 37° under standardconditions. The reaction was stopped by phenol extraction. Thelinearised vector was then precipitated with ethanol, washed andresuspended in water.

C. Cloning of the pTOM6 HinfI fragment into pDH51

The isolated HinfI fragment from pTOM6 (A) and linearised vector (B)were ligated overnight under standard conditions. The ligation mix wasused to transform competent TG2 cells. The transformation mix was platedonto ampicillin-containing plates. Clones were selected, DNA isolatedand analysed by digestion with BamHI and HindIII restriction enzymes.Plasmids were identified, and were named pDHC1 and pDHC4. pDHC1 containsthe HinfI fragment in the antisense orientation; pDHC4 contains theHinfI fragment in the sense orientation.

EXAMPLE 3 Construction of Plasmid pCB1

A. Isolation of a PG promoter fragment

Genomic clones were isolated from a partial Sau3A library of Ailsa Craigtomato DNA cloned into EMBL3 (Bird et al, in preparation). PG cloneswere isolated from the genomic library by screening with both thecomplete pTOM6 cDNA insert, and the isolated 5′ Pst1/HindIII fragmentfrom pTOM6 (Grierson et al, NAR 1986). Several overlapping clones wereisolated and the transcription start site of the PG gene located by S1mapping experiments (Bird et al in preparation 1987). The PG promotercan be located on a 1.6 Kb HindIII fragment which also contains part ofthe PG coding information.

B. Insertion of a SpeI site into the PG promoter fragment

In order to be able to manipulate the PG promoter sequence conveniently(ie. the DNA 5′ to the transcription start) a Spel site was introducedby site directed mutagenesis using standard protocols. The HindIIIfragment was isolated from genomic clone gTOM23 (NCIB Accession No12373) and cloned into the HindIII site of (commercially availablevector) M13 mp19. After annealing with a suitable mismatch primer andextension using DNA polymerase, the mixture was transformed intocompetent TG2 cells. Phages were plated and duplicated filters wereprepared for hybridisation to the labelled mismatch primer. Putativeclones were identified by hybridisation under increasingly stringentconditions, isolated and the generation of the Spel site was determinedby direct DNA sequence analysis. The promoter fragment was isolated fromone isolate by restriction with Spel and HindIII. This fragment was thencloned into pUC19 (commercially available plasmid) cut with HindIII andXbaI. The promoter fragment was then transferred into Bin19 (Bevan,Nucleic Acid Research, 1984, 12, 8711-8721) cut with BamH1 and HindIII.The resulting plasmid was called pCB1.

EXAMPLE 4 Construction of Plasmid pJR2

A. Isolation of the PG promoter fragment from pCB1

5 μg of pCB1 (prepared as in Example 2) was cut with HindIII for 2 hoursat 37° C. The mixture was phenol/chloroform extracted and DNAprecipitated with ethanol. After re-suspension in water the cohesiveends were filled in using DNA polymerase under standard conditions atroom temperature for 15 minutes. The polymerase was inactivated byheating to 65° C. for 15 minutes. The DNA was then treated with BamH1for 2 hours at 37° C. The PG promoter fragment was then byelectroelution isolated by agarose gel electrophoresis as aHindIII/BamH1 1.45 Kb fragment.

B. Preparation of pJR1 for insertion of the PG promoter fragment

5μg of pJR1 (constructed in Example 6) was cut with NcoI for 2 hours at37° C. under standard conditions. The DNA was purified by extractionwith phenol and chloroform. The cohesive ends were filled in using DNApolymerase I Klenow fragment A for 15 minutes at room temperature. Thevolume was increased and BamHI added. The mixture was incubated for 2hours at 37° C. The mixture was then fractionated on agarose gels, andthe large fragment of approximately 3 kb isolated by electroelution.

C. Cloning of the PG promoter into the large fragment from pJR1

pJR1 prepared as in (B) above was ligated with the PG promoter fragmentprepared in (A) under standard conditions for 24 hours at 16° C. Analiquot of the ligation mixture was used to transform competent TG2cells. Aliquots of the transformation mixture were plated onto L platescontaining ampicillin and Xgal. Colonies were picked and examined forthe presence of the PG promoter DNA by electrophoresis on agarose gelsin order to detect an increase in the size of the vector and by directDNA sequence determination. Plasmids containing the PG promoter werecalled pJR2.

Construction of Antisense and Sense PG Vectors

A series of antisense and sense vectors containing different portions ofthe PG cDNA and PG gene were constructed for use in regeneratingtransgenic plants. The vectors produced are summarised in Table 1. Thevectors constructed are based on PJR1 and pJR2. DNA fragments have beeninserted into these vectors both into the antisense (A) and sense (B)orientations. Expression cassettes contained in these vectors were thentransferred to Bin19 (Bevan (1984) Nucleic Acid Research, 12, 8711-8721)for transformation of tomato plants.

TABLE 1 Name of Name of Vectors antisense sense PG fragment based onvector vector (see Figure 2) pJR1 pJR16A pJR16S 740 bp HinfI pJR36ApJR36S fragment a pJR56A pJR56S fragment b pJR76A pJR76S fragment c pJR2pJR26A pJR26S 740 bp HinfI pJR46A pJR46S fragment a pJR66A PJR66Sfragment b pJR86A pJR86S fragment c

EXAMPLE 5 Construction of PG Vector pJR16S

A. Preparation of pJR1

1 μg pJR1 (from Example 1) was cut with KpnI and PstI at 37° C. for 2hours. The reaction was stopped by extraction with phenol andchloroform. The DNA was precipitated with ethanol, washed and dried. Thevector was resuspended in 20μl TE.

B. Isolation of a 740 bp PG sense fragment

5 μg pDHC4 was cut with KpnI and PstI at 37° C. for 2 hours understandard conditions. The 740 bp fragment produced was isolated afteragarose gel electrophoresis by electroelution. The fragment wasextracted with phenol and chloroform and precipitated with ethanol. Thefragment was then suspended in 10 μl TE.

C. Ligation of the PG sense fragment to pJR1

The products of (B) were ligated at 16° C. for 24 hours under standardconditions. The ligation mix was used to transform competent TG2 cellsand the mixture was plated onto ampicillin containing plates. Singlecolonies were grown up to prepare plasmid DNA. The DNA was analysed forthe presence of a 900 bp HindIII fragment. A suitable clone wasidentified and called pJR16S.

EXAMPLE 6 Transfer of pJR16S to Bin 19

A. Isolation of the 1600 bp expression cassettes

pJR16S was cut with EcoRI at 37° C. for 2 hours under standardconditions. An aliquot of the reaction mixture was separated by agarosegel electrophoresis to check that the reaction had gone to completion.It was then heated to 65° C. for 15 minutes in order to inactivate theenzyme. The DNA was then cut partially with a small amount of HindIII inorder to give all the possible EcoRI/HindIII partial digestionfragments. The EcoRI-HindIII fragment of approximately 1600 bpconsisting of the 35S CaMV promoter, the PG insert sequences and Nos 3′end (expression cassette) was isolated after agarose gel electrophoresisby electroelution. The fragment was extracted with phenol andchloroform, and precipitated with ethanol. The fragment was washed,dried and resuspended in 10 μl TE.

B. Preparation of Bin 19 for cloning

5 μg Bin19 DNA was cut with EcoRI and HindIII at 37° for 2 hours understandard conditions. The reaction was stopped by phenol and chloroformextraction, and DNA was precipitated with ethanol. The vector preparedin this fashion was resuspended in 20 μl TE.

C. Ligation of Bin19 to PG Expression Cassettes

The products of (A) and (B) were set up for ligation. Aliquots of the PGsense cassette were ligated to Bin19 at 16° C. for 24 hours understandard conditions. The ligation mixes were used to transform competentTG2 cells which were plated on L agar containing Kanamycin and Xgal.Recombinant colonies were identified by their white colour. A number ofthese were picked from each ligation reaction and used to prepareplasmid DNA. The DNA was analysed for the relevant restriction patternby cutting with EcoRI and HindIII.

EXAMPLE 7 Construction of PG Vector pJR26S

A. Preparation of pJR2

1 μg pJR2 (from Example 4) was cut with HindIII and PstI at 37° C. for 2hours under standard conditions. The reaction was terminated byextraction with phenol and chloroform and precipitated with ethanol. Thepurified vector was resuspended in 20 μl TE.

B. Isolation of the 740 bp PG fragment

5 μg pDHC4 were cut with KpnI at 37° C. for 2 hours under standardconditions. The cohesive ends of the DNA were filled in with T4 DNApolymerase. The reaction was stopped by heating to 65° C. for 15minutes. The DNA was then also cut with PstI. The resulting 740 bpfragment was isolated after agarose gel electrophoresis byelectroelution. The fragment was extracted with phenol and chloroformand precipitated with ethanol. It was then resuspended in 10 μl TE.

C. Ligation of the PG Sense Fragment to pJR2

The products of (A) and (B) above were ligated at 16° C. for 24 hoursunder standard conditions. The ligation mix was used to transformcompetent TG2 cells, plated onto ampicillin containing plates andincubated at 37° C. overnight. Transformed single colonies were grown upand plasmid DNA was prepared. The DNA was analysed for the presence of a1.6 Kb EcoRI-HindIII fragment. A clone was identified and called pJR26S.

EXAMPLE 8 Transfer of pJR26S to Bin19

A procedure essentially the same as described above in Example 6 wasused to subclone the 2.6 Kb EcoRI-HindIII partial fragment from pJR26Sinto Bin19 cut with EcoR1 and HindIII. Recombinants were identified bytheir white colour reaction after plating onto L-agar plates containingkanamycin and Xgal. Recombinants were characterised by restrictiondigestion with EcoR1 and HindIII.

EXAMPLE 9 Construction of Vectors pJR36S (Fragment a) and pJR46S(Fragment b)

A. Isolation of fragments (a) and (b)

5 μg pDHC4 (Example 2) was cut with KpnI and BamHI at 37° C. for 2 hoursunder standard conditions. The 500 bp fragment was isolated afteragarose gel electrophoresis by electroelution, extracted with phenol,chloroform and resuspended in 20 μl TE. The KpnI-BamHI fragment was thencut with HindIII. The cohesive ends of the fragment were filled with T4DNA polymerase. The resulting fragments: a) 199 bp HindIII-KpnI (bluntended) and b) 275 bp HindIII-BamNI (blunt ended) were isolated afteragarose gel electrophoresis by electroelution, extraction with phenoland chloroform, and resuspended in 10 μl TE.

B. Preparation of pJR1

1 μg pJR1 (from Example 1) was cut with SmaI at 37° C. for 2 hours understandard conditions. The reaction was stopped by extraction with phenoland chloroform, and precipitated with ethanol. The vector was thenresuspended in 20 μl TE.

C. Ligation of fragment (a) into pJR1

pJR36S

Fragment (a) from (A) above was ligated to SmaI cut pJR1 (from (B)above) at 16° C. for 24 hours under standard conditions. The ligationmixture was used to transform competent TG2 cells which were then platedonto ampicillin-containing plates. Transformed colonies were grown upand used for plasmid DNA preparation. EcoRI/PstI double digestsidentified those clones containing fragment (a) inserts. The EcoRI-PstIinserts of these clones were isolated and subcloned into M13 mp8 whichhad been cut with EcoRI and Pst I. DNA sequence analysis was carried outin order to ascertain the orientation of the insert (a). Clones obtainedfrom this experiment were called pJR36A and pJR36S, according to theorientation of the insert.

D. Ligation of fragment (b) into pJR1

pJR56S

Fragment (b) from (A) above was ligated to SmaI cut pJR1, from (B)above, at 16° C. for 24 hours under standard conditions. The ligationmixture was used to transform competent TG2 cells which were then platedonto ampicillin containing plates. Transformed colonies were grown upand used for plasmid DNA preparation. EcoRI/PstI double digestidentified those clones containing fragment (b) inserts. The EcoRI-PstIinserts of these clones were isolated and subcloned into M13 mp8 whichhad been cut with EcoRI and PstI. DNA sequence analysis was carried outin order to ascertain the orientation of insert (b). Clones obtainedfrom this experiment were called pJR56A and pJR56S, according to theorientation of the insert.

EXAMPLE 10 Transfer of pJR36S and pJR56S to Bin19

A. Preparation of expression cassettes containing fragments (a) and (b)in pJR1

5μg each of pJR36S and pJR56S were cut separately with EcoRI and HindIIIat 37° C. for 2 hours under standard conditions. The resulting twofragments containing la) 930 bp and (b) 1000 bp were isolated separatelyafter electrophoresis on agarose gels by electroelution. The fragmentswere extracted with phenol and chloroform, and precipitated withethanol. The two fragments were then resuspended in 10 μl TE.

B. Preparation of Bin19

Bin19 was cut with EcoRI and HindIII for 2 hours at 37° C. understandard conditions. The reaction was stopped by addition of phenol andchloroform. After extraction the DNA was precipitated with ethanol, andresuspended in 2 μl TE.

C. Ligation of the fragments to Bin19

The two EcoRI-HindIII fragments isolated in A were set up for separateligation reactions using Bin19 prepared as described in B under standardconditions. The ligation mixtures were used to transform competent TG2cells which were plated onto L agar containing kanamycin and Xgal. Afterincubation overnight, recombinant colonies were identified by theirwhite colour. A number of the clones were picked from each separateligation and were used to prepare DNA. The DNA's were analysed for thepresence of a EcoRI-HindIII fragment of the appropriate size for theinsertion of the expression cassettes to Bin19.

EXAMPLE 11 Construction of pJR46S (Fragment a) and pJR66S (Fragment b)

A. Preparation of pJR2

1 μg pJR2 (from Example 3) was cut with HincII at 37° C. for 2 hoursunder standard conditions. The reaction was terminated by extractionwith phenol and chloroform. The vector was precipitated with ethanol,washed and resuspended in 20 μ1 TE.

pJR46S

B. Ligation of PG fragment (a) to pJR2

Fragment (a) from Example 9(A) above was ligated to HincII cut pJR2 from(A) above at 16° C. for 24 hours under standard conditions. The ligationmixture was used to transform competent TG2 cells which were then platedonto ampicillin containing plates. Transformants were picked, grown upand used to prepare plasmid DNA. Plasmid DNA was cut with both EcoRI andPstI. DNA from clones which contained inserts were restricted with EcoRIand Pst I. The EcoRI-PstI inserts were isolated after agarose gelelectrophoresis by electroelution and subcloned into M13mp8 which hadbeen cut with EcoRI and PstI. DNA sequence analysis was used toascertain the orientation of the inserts (a). Clones were obtained fromthis experiment were called pJR46A and pJR46S, according to theorientation of the insert.

pJR66S

C. Ligation of PG fragment (b) to pJR2

Fragment (b) from Example 9 (A) was ligated to HincII cut, from A above,separately at 16° C. for 24 hours under standard conditions. Theligation mixture was used to transform competent TG2 cells which werethen plated onto ampicillin containing plates. Transformants werepicked, grown up and used to prepare plasmid DNA. Plasmid DNA was cutwith both EcoRI and PstI. DNA from clones which contained inserts wererestricted with EcoRI and PstI. The EcoRI-PstI inserts were isolatedafter agarose gel electrophoresis by electroelution and subcloned intoN13 mp8 which had been cut with EcoRI and PstI. DNA sequence analysiswas used to ascertain the orientation of the inserts (b). Clones wereobtained from this experiment were called pJR66A and pJR66S, accordingto the orientation of the insert.

EXAMPLE 12

Transfer of pJR46S and pJR66S to Bin19

A. Preparation of expression cassettes containing fragments (a) and (b)in pJR2

5 μg of each of pJR46S and pJR66S were cut separately with EcoRI andHindIII at 37° C. for 2 hours under standard conditions. The resultingtwo fragments of approximately 2.5 kb were isolated separately after gelelectrophoresis by electroelution. The fragments were extracted withphenol and chloroform, and precipitated with ethanol. The two fragmentswere then resuspended in 10 μl TE.

B. Ligation of expression cassettes into Bin19

Aliquots containing the two fragments from (A) were ligated to Bin19 DNAprepared as described in Example 15 (B) in separate ligation reactionsunder standard conditions. The ligation mixtures were used to transformcompetent TG2 cells. The transformation mixture was plated onto L-agarplates containing kanamycin and Xgal. After overnight incubationrecombinant colonies were identified by their white colour. A number ofclones for the separate experiments were picked and DNA was prepared.The DNAs were analysed for the presence of the appropriateEcorRI-HindIII fragments.

EXAMPLE 13 Construction of PG Vector pJR76S

A. Isolation of fragment (c)

10 μg gTOM 23 (a genomic clone containing the PG gene, NCIB No 12373)was cut with HindIII and BamH1. The 1.98 Kb fragment was isolated afteragarose gel electrophoresis by electroelution. The cohesive ends of thefragment were filled in with T4 DNA polymerase.

B. Ligation of fragment (c) to pJR1

The products from (A) above and Example 9(B) (ie. pJR1 cut with SmaI)were ligated at 16° C. for 24 hours under standard conditions and themixture used to transform competent TG2 cells which were then platedonto plates containing ampicillin. Transformed colonies were grown upand used to prepare plasmid DNA. The DNA was cut with EcoRI and theorientation of the insert determined from the pattern of fragmentsobtained. The clones were called pJR76A and pJR76S according to theorientation of the insert.

EXAMPLE 14 Transfer of Vector pJR76S to Bin19

A. Preparation of expression cassette from pJR76S

5 μg of the clone was cut with HindIII at 37° C. for 2 hours understandard conditions. The enzyme was inactivated by heating the reactionmixture to 70° C. for 15 minutes. EcoRI was then added in concentrationnecessary to give partial restriction. The reaction was stopped by theaddition of phenol and chloroform. The required 2.71 Kb EcoRI-HindIIIfragments was isolated after agarose gel electrophoresis byelectroelution. The fragment was extracted with phenol and chloroformand precipitated with ethanol, and then resuspended in 10 μl TE.

B. Ligation of the expression cassette to Bin19

The fragment from pJR76S prepared in (A) was ligated to Bin19 (preparedas described in Example 6B). The ligation mixture was used to transformcompetent TG2 cells. The transformation mix was plated onto L platescontaining kanamycin and Xgal. Recombinant plasmids were identified bytheir white colour. DNA was prepared from a number of these and analysedfor the presence of the required EcoRI-HindIII fragments.

EXAMPLE 15 Construction of PG Vector pJR86S

A. Ligation of PG fragment (c) to pJR2

The products of Example 13 (A) (fragment c) and Example 11 (A) (ie pJR2cut with HincII) were ligated at 16° C. for 24 hours under standardconditions and the mixture used to transform competent TG2 cells whichwere then plated on plates containing ampicillin. Transformed colonieswere grown up and used to prepare plasmid DNA. The orientation of theinsert was deduced using the EcoRI restriction pattern. These cloneswere called pJR86A and pJR86S, according to the orientation of theinsert.

EXAMPLE 16 Transfer of Vector pJR86S into Bin19

This Example was carried out essentially as described in Example 14, ie.the vectors was cut with HindIII under conditions of partialrestriction, which was then followed by restriction with EcoRI. Theresulting 3.63 Kb fragment was isolated and cloned into Bin19.

All constructs in Bin19 were intended for use in separate triparentalmating experiments to allow transfer to Agrobacterium, and from there totomato plants.

Inhibition of Pectin Esterase

In addition to polygalacturonase, pectin esterase (PE) has beenimplicated in softening of the tomato fruit. A ripe tomato fruit cDNAlibrary was screened with mixed oligonucleotide probes designed from thepublished amino acid sequence of PE. One clone, pPE1, (NCIB Accession No12568) has been isolated and characterised. The complete sequence ofthis cDNA clone is shown in FIG. 3 of European Patent Specification271988 (U.S. Ser. No. 07/119614).

We have used fragments of the cDNA to construct antisense and sensevectors. These are summarised in Table 2.

TABLE 2 Name of Vectors Name of anti- sense based on sense vector vectorFragment pJR1 pJR101A pJR101S 420 bp PstI pJR111A pJR111S 351 bp BbvIpJR2 pJR102A pJR102S 420 bp PstI pJR112A pJR112S 351 bp BbvI

Construction of PE Vectors EXAMPLE 17 Preparation of pJR101S

A. Isolation of a 420 bp fragment from pPE1

Plasmid pPE1 was cut with PstI at 37° C. for 2 hours under standardconditions. The 420 bp PstI fragment was isolated after agarose gelelectrophoresis by electroelution, extracted with phenol and chloroformand precipitated with ethanol. The DNA was then resuspended in 10 μl TE.

B. Preparation of pJR1

pJR1 (Example 1) was cut with PstI at 37° C. for 2 hours under standardconditions. The reaction was stopped by the addition of phenol,precipitated with ethanol and resuspended in 20 μl TE.

C. Ligation of PE fragment to PJR1

The products of steps (A) and (B) above were ligated under standardconditions and the ligation mixture was used to transform competent TG2cells. The transformation mix was subsequently plated onto ampicillincontaining plates and incubated at 37° C. overnight. Transformedcolonies were grown up and used to prepare plasmid DNA. Clones wereidentified which gave 420bp fragment on digestion with PstI. The 650 bpBamHI-HindIII fragments from these clones were isolated after agarosegel electrophoresis by electroelution and cloned into M13mp8. Theorientation of the PstI insert was determined by sequence analysis.Clones identified were named pJR101A and pJR101S according to theorientation of the insert.

EXAMPLE 18 Transfer of Vector pJR101S to Bin19

A. Isolation of a 1.2 Kb EcoRI-HindIII fragment

Plasmid pJR101S was cut with EcoRI and HindIII at 37° C. for 2 hoursunder standard conditions. The resulting 1.2 Kb fragment was isolatedafter gel electrophoresis from agarose gel by electroelution. The DNAwas then extracted with phenol and chloroform, precipitated withethanol, and resuspended in 20 μl TE.

B. Preparation of Bin19

Bin19 was cut with EcoRI and HindIII for 2 hours at 37° C. understandard conditions. The enzymes were removed by phenol extraction andthe vector precipitated with ethanol. The DNA was then resuspended inwater.

C. Ligation of the PE expression cassette to Bin19

Aliquots of the products of reactions A and B were ligated for 16 hoursat 16° C. under standard conditions. The ligation mix was used totransform competent TG2 cells. The transformation mix was plated ontoplates containing kanamycin. DNA was picked from individual clones andanalysed for the presence of the 1.2 Kb EcoR1-HindIII fragment.

EXAMPLE 19 Construction of Vector pJR102S

The construction of this vector followed the construction of pJR101S(Example 17) except that the 420bp Pst1 fragment was inserted into pJR2(from Example 3).

EXAMPLE 20 Transfer of Vector pJR102S to Bin19

Transfer of the PE expression cassette to Bin19 was carried out asdescribed in Example 18 for the transfer of pJR102S into Bin19.

EXAMPLE 21 Construction of Vector pJR111S

A. Isolation of a 351 bp fragment from pPE1

Plasmid pPE1 was cut with BbvI at 37° C. for 2 hours under standardconditions and the cohesive ends filled using T4 polymerase. The 351 bpfragment was isolated after agarose gel electrophoresis byelectroelution, extracted with phenol and chloroform and precipitatedwith ethanol. It was then resuspended in 10 μl TE.

B. Preparation of pJR1

1 μg pJR1 (from Example 1) was cut with SmaI for 2 hours at 37° C. understandard conditions. The reaction was terminated by the addition ofphenol and chloroform. After extraction the DNA was precipitated withethanol, and resuspended in 10 μl TE.

C. Ligation of the PE fragment to pJR1

The products of (A) and (B) were ligated at 16° C. for 24 hours understandard conditions. The ligation mix was used to transform competentE.coli TG2 cells. The transformation mix was plated ontoampicillin-containing plates. Single colonies were grown up and analysedfor the presence of a 900 bp EcoR1-PstI fragment. This fragment wasisolated by electroelution after agarose gel electrophoresis and clonedinto M13 mp8 (commercially available vector). The orientation of thefragment was determined by DNA sequence analysis.

EXAMPLE 22 Transfer of Vector pJR111S to Bin19

A. Isolation of the 1.1 Kb EcoR1-HindIII fragment

Plasmid pJR111S was cut with EcoR1 and HindIII at 37° C. for 2 hoursunder standard conditions. The 1.1 Kb fragment was isolated afteragarose gel electrophoresis by electroelution. It was extracted withphenol and chloroform, precipitated with ethanol and resuspended in 20μ1 TE.

B. Ligation of the PE expression cassette into Bin19

Aliquots of the products of (A) and Example 18 (B) were ligated at 16°C. for 24 hours. The ligation mixtures were used to transform competentE.coli TG2cells. The transformation mix was plated onto platescontaining kanamycin. Single colonies were used for DNA extraction andclones identified by the presence of the 1.1 Kb EcoR1-HindIII fragment.

EXAMPLE 23 Construction of Vector pJR112S

Construction of this vector followed the procedure in Example 34 for theconstruction of pJR111S except that the 351 bp BbvI fragment wasinserted into pJR2 from Example 4, rather than pJR1.

EXAMPLE 24 Transfer of Vector pJR112S to Bin19

Transfer of the PE expression cassette from pJR112S into Bin19 followedthe protocol described in Example 22.

EXAMPLE 25 Transformation of Tomato Stem Explants

A. Transfer of Bin19 vectors to Agrobacterium

The recombinant vectors prepared in Example 6 were mobilised from E.coli(TG-2) to Agrobacterium tumefaciens (LBA4404) (Hoekma A, Hirsch P R,Hooykaas P J J and Schilperoort R A, 1983, Nature.303 ppl 79-180) in atriparental mating on L-plates with E.coli (HB101) harbouring pRK2013(Ditta G. et al, 1980 PNAS,USA, Vol 77, pp7347-7351) Transconjugantswere selected on minimal medium containing kanamycin (50 μ/cm³) andstreptomycin (500 μ/cm³).

B. Preparation of Agrobacteria for transformation

L-Broth (5 cm³) containing kanamycin at 50 μ/cm³ was inoculated with asingle bacterial colony. The culture was grown overnight at 30° C. withshaking at 150 r.p.m. This culture (500 μ) was inoculated into L-Brothcontaining kanamycin (50 μ/cm³) and grown as before. Immediately beforeuse the Agrobacteria were pelleted by spinning at 3000 r.p.m. for 5minutes and resuspended in an equal volume of liquid Murashige and Skoog(MS) medium.

C. Preparation of plant tissue for transformation

Feeder plates were prepared in 9 cm diameter petri dishes as follows.Solid MS medium supplemented with 5 μM zeatin riboside and 3 μM IAAaspartic acid was overlaid with Nicotiana tabacum var Samsun suspensionculture (1 cm³ ). One 9 cm and one 7 cm filter paper discs were placedon the surface. Hypocotyls from 4 week old tomato seedlings grown on MSmedium were excised and placed on feeder plates. The plates were sealedwith Nescofilm and incubated overnight in the plant growth room (26° C.under bright fluorescent light).

D. Transformation Protocol Hypocotyls from feeder plates were placed inthe Agrobacteria suspension in 12 cm diameter petri dishes and cut intoapproximately 1 cm lengths, removing all leaf and cotyledon axes. After20 minutes the hypocotyl segments were returned to the feeder plateswhich were sealed and replaced in the growth room. After 48 hoursincubation in the growth room the plant material was transferred to MSmedium supplemented with 5 μM zeatin riboside, 3 μM IAA aspartic acid,500 μg/cm³ carbenicillin and 50 μg/cm³ kanamycin in petri dishes. Thepetri dishes were sealed and returned to the growth room.

From six weeks after inoculation with Agrobacterium, shoots were removedfrom the explants and placed on MS medium supplemented withcarbenicillin (200 μ/cm³) for rooting. Transformed plants rooted 1-2weeks after transfer.

These plants were then grown in tissue culture for a number of weeksbefore being transferred to pots. These plants were then grown in growthrooms or greenhouses as appropriate.

EXAMPLE 26 Molecular Analysis of Transformed Plants

Leaf material from plants which had been transformed using the vectorpJR16S (see Example 5) was analysed to detect the expression vector bySouthern hybridisation or polymerase chain reaction (PCR). Leaf materialwas used to determine the expression of RNA encoding the PG fragment.

EXAMPLE 27 Analysis of PG Levels in Fruit From Plants Transformed WithpJR16S

Transformed tomato plants identified in Example 26 as expressing RNAencoding the PG fragment from pJR16S were grown to maturity. The fruitfrom each plant were analysed using standard techniques for the presenceof PG enzyme. Results for 13 such plants are shown in Table 3. Datashown here identify clearly plants in which PG activity has been reducedto 5% or less of the corresponding activity in untransformed controltomatoes. “Breaker” in the Table denotes the time at which the tomatoesbegin to change colour from green to orange. The plants in this Exampleare believed to be heterozygous for the pJR16S construct: by selectionand crossing plants that are homozygous for this construct can readilybe obtained. These are likely to show even further reduction in PGactivity.

TABLE 3 Levels of PG found in plants transformed with expression vectorpJR16S PG activity at Transformed breaker + 10 days Plant (% of normal)GR174 51 GR242 65 GR241 77 GR240 25 GR248 13 GR249  4 GR250 74 GR254 16GR255 75 GR263  9 GR285 10 GR267 25 GR270 61

Deposit of Microorganisms

The specification refers to four microorganisms that have beendeposited. These are shown below. All four were deposited at theNational Collections of Industrial and Marine Bacteria Limited (NCIMB)of Aberdeen, Scotland (current address 23 St Machar Drive, Aberdeen AB21RY).

Strain NCIMB designation Date of Deposit Accession No. E. coli C600(pTOM6) Nov. 7, 1986 12351 E. coli DH5α (pWRECK2- Nov. 7, 1986 12352CAMV-CAT-1) E. coli K803 (gTOM23) Dec. 5, 1986 12373 E. coli TG2 (pPE1)Oct. 20, 1987 12568

Reference to Related Applications

This application is a continuation-in-part of U.S. application Ser. No07/119,614 filed Nov. 12 1987, which claimed priority from Britishapplication No 8626879 filed Nov. 11, 1986. The full disclosure of U.S.Ser. No. 07/119,614 is incorporated herein by reference.

We claim:
 1. A process for modifying the production of a target geneproduct in a plant cell which comprises transforming the plant cell witha construct comprising a recombinant DNA sequence coding for only partof the target gene product wherein said target gene product is a fruitripening enzyme.
 2. Process as claimed in claim 1 in which thefruit-ripening enzyme is polygalacturonase.
 3. The process of claim 1wherein the gene product is polygalacturonase or pectinesterase, saidrecombinant DNA sequence being shorter than the sequence encodingpolygalacturonase or pectinesterase but sufficient to inhibit theexpression of said polygalacturonase or pectinesterase.
 4. The processof claim 3 wherein the plant cells are tomato plant cells.
 5. Theprocess of claim 1 wherein the enzyme is pectinesterase, galactosidase,glucanase, xylanase or cellulase.
 6. The process of claim 5 wherein therecombinant DNA sequence comprises at least 50 bases of the geneencoding said enzyme.
 7. The process of claim 6 wherein the DNA sequencecomprises up to 1000 bases of said gene.
 8. The process of claim 1wherein the plant cells are tomato cells and the construct is selectedfrom the group consisting of pJR36S, pJR56S, pJR76S, pJR26S, pJR46S,pJR66S, pJR86S, pJR101S, pJR111S, pJR102S and pJR112S.